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OBJECTIVE@#Critical effective constituents were identified from Bufei Yishen formula (BYF), a traditional herbal compound and combined as effective-constituent compatibility (ECC) of BYF I, which may have potential bioactive equivalence to BYF.@*METHODS@#The active constituents of BYF were identified using four cellular models and categorised into Groups 1 (Bufeiqi), 2 (Bushen), 3 (Huatan) and 4 (Huoxue) according to Chinese medicinal theory. An orthogonal design and a combination method were used to determine the optimal ratios of effective constituents in each group and the ratios of "Groups 1 to 4" according to their pharmacological activity. We also comprehensively assessed bioactive equivalence between the BYF and the ECC of BYF I in a rat model of chronic obstructive pulmonary disease (COPD).@*RESULTS@#We identified 12 active constituents in BYF. The numbers of constituents in Groups 1 to 4 were 3, 2, 5 and 2, respectively. We identified the optimal ratios of effective constituents within each group. In Group 1, total ginsenosides:Astragalus polysaccharide:astragaloside IV ratio was 9:5:2. In Group 2, icariin:schisandrin B ratio was 100:12.5. In Group 3, nobiletin:hesperidin:peimine:peiminine:kaempferol ratio was 4:30:6.25:0:0. In Group 4, paeoniflorin:paeonol ratio was 4:1. An orthogonal design was then used to establish the optimal ratios of Group 1, Group 2, Group 3 and Group 4 in ECC of BYF I. The ratio for total ginsenosides:Astragalus polysaccharide:astragaloside IV:icariin:schisandrin B:nobiletin:hesperidin:peimine:paeoniflorin:paeonol was determined to be 22.5:12.5:5:100:12.5:4:30:6.25:25:6.25. A comprehensive evaluation confirmed that ECC of BYF I presented with bioactive equivalence to the original BYF.@*CONCLUSION@#Based on the ECC of traditional Chinese medicine formula method, the effective constituents of BYF were identified and combined in a fixed ratio as ECC of BYF I that was as effective as BYF itself in treating rats with COPD.
ABSTRACT
To establish high performance liquid chromatography(HPLC) fingerprints for crude and processed Ligustri Lucidi Fructus,and to evaluate their quality through the similarity calculation and chemical pattern recognition. The separation was performed with Syncronis C_(18) column(4.6 mm × 250 mm, 5 μm), with acetonitrile(A) and 0.1% phosphoric acid solution(B) as the mobile phase for gradient elution, and a detection wavelength of 280 nm. HPLC was used to detect 22 batches of crude and processed Ligustri Lucidi Fructus,and the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(2012 Edition) was used to evaluate the similarity among 22 batches. The research on pattern recognition was conducted with cluster analysis(CA), principal component analysis(PCA), and partial least squares discriminate analysis(PLS-DA). HPLC fingerprints of crude and processed Ligustri Lucidi Fructus were established, with similarity ranging from 0.9 to 1.0. The crude and processed Ligustri Lucidi Fructus can be obviously distinguished by using CA, PCA and PLS-DA. According to the results of PLS-DA,11 constituents including hydroxytyrosol, tyrosol, specnuezhenide and oleuropein were the main marker components leading to the difference. The established fingerprint method is stable and reliable, and can provide method basis for quality control of crude and processed Ligustri Lucidi Fructus. Chemical pattern recognition is proved to be helpful in comprehensive quality control and evaluation of Ligustri Lucidi Fructus before and after the process.
Subject(s)
Chromatography, High Pressure Liquid , Drugs, Chinese Herbal , Fruit , Ligustrum , Medicine, Chinese TraditionalABSTRACT
Objective To develop a simple, sensitive, and precise method for simultaneous determination of 10 anthraquinones in Rhubarb. Methods HPLC-Q-HR/MS was employed for simultaneous quantification of free anthraquinones (aloe-emodin, emodin, chrysophanol, physcion, and rhein) and their glycosides. Chromatographic analysis was performed on an XBridge™ C18 column (2.1 mm × 150 mm, 5 µm) with mobile phases consisting of 3 mmol/L ammonium acetate (A) and methanol (B) at a flow rate of 0.3 mL/min. Results All calibration curves exhibited good linear relationship (R2 > 0.999). The limits of detection (LOD) and quantification (LOQ) were in the range of 0.39-2.97 ng/mL and 0.56-8.90 ng/mL, respectively. The overall intra- and inter-day precisions of analytes presented as relative standard deviations (RSDs) were less than 2.79%. Relative recoveries varied between 97.83% and 104.28%. The validated method was applied to assess the quality of Rhubarb collected from different regions of China. Results showed that chrysophanol and rhein-8-O-β-D-glucoside was the largest portion of free anthraquinones and anthraquinone glycosides in Rhubarb, respectively. The total content of anthraquinones was higher in Rhubarbs from Sichuan, Qinghai, Yunnan, and Gansu provinces than that in those from Shandong and Henan provinces, while no significant variability existed in different regions of the same province. Conclusion HPLC-Q-HR/MS method is accurate and reliable for simultaneous quantification of above free anthraquinones and their glycosides in Rhubarb and can be applied to standardize the quality of Rhubarb and its quality control in different regions.
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Objective: To establish a method for determination of emodin in rat plasma by HPLC/Q-Exactive HR/MS, and to study the pharmacokinetics of emodin in normal rats and cerebral ischemia rats. Methods: The plasma concentration of emodin was determined by HPLC/Q-Exactive HRMS method with internal standard method. Emodin was eluted on a XBridgeTM C18 (150 mm × 2.1 mm, 5 μm) column with temperature at 30 ℃. The mobile phase consisted of 3 mmol/L ammonium acetate and methanol, with a gradient program as follows: 0~2 min (30% methanol), 2-10 min (30%-60% methanol), 10-13 min (60%-30% methanol). The flow rate was 0.3 mL/min, and the injection volume was 5 μL. MS experiments were coupled with the HPLC via HESI source operated in negative ionization full-scan mode. The pharmacokinetic parameters were calculated by the software of DAS 3.0. Results: The main pharmacokinetic parameters of emodin in normal rats and cerebral ischemia rats were as follows: AUC0-∞ were (605.63 ± 163.66) and (1 107.78 ± 191.11) ng∙h/mL, Cmax were (81.96 ± 20.72) and (91.65 ± 16.82) ng/mL, VZ/F were (851.03 ± 97.30) and (1 051.87 ± 119.88) L/kg, t1/2 were (10.31 ± 1.61) and (23.13 ± 3.56) h, tmax were (0.75 ± 0.22) and (0.75 ± 0.16) h. Conclusion: The method is simple, accurate, fast, sensitive, and suitable for the pharmacokinetic study of emodin in rats.
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Rhubarb is widely used in the treatment of obstipation, gastrointestinal indigestion and other diseases in China and other Asian countries for thousands of years. Anthraquinones are the major group of polyphenol constituents including aloe-emodin, rhein, emodin, chrysophanol and physcion. In order to evaluate the pharmacokinetics of five rhubarb anthraquinones, a high-performance liquid chromatography with fluorescence detection [HPLC-FLD] method for simultaneous determination of aloe-emodin, rhein, emodin, chrysophanol and physcion in dog plasma was established. Solid phase extraction [SPE] was applied to the extraction and purification of samples. The calibration curves of five anthraquinones showed good linearity with r greater than 0.9925. The average extraction recoveries, examined at three concentration levels, carried from 92.1% to 102.3%, and the accuracies ranged from 87.7% to 102.5% with precision [RSD] <10%. The pharmacokinetic paremeters of five anthraquinones were investigated systematically after orally administration the rhubarb extract. Five anthraquinones were rapidly absorbed and T[max] for aloe-emodin, rhein, emodin, chrysophanol and physcion was at 0.75, 1.50, 0.75, 1.0 and 2.0 h respectively. The C[max] of five anthraquinones was 0.031, 3.39, 0.27, 0.036 and 0.032 micro g/mL while the AUC of five anthraquinones was 0.35 +/- 0.058, 32.22 +/- 8.29, 2.97 +/- 0.66, 0.43 +/- 0.10 and 0.41 +/- 0.12 mg h/L, respectively